The retinal pigment epithelium (RPE) is a highly metabolically active, post-mitotic tissue, which performs numerous functions that are indispensable for vision. A key function of the RPE is the daily phagocytosis and degradation of shed photoreceptor outer segments. These two features - high metabolism and circadian digestion of phagocytosed outer segments - place a heavy burden on cellular clearance mechanisms in the RPE. Inefficient disposal of debris by the RPE promotes the accumulation of insoluble aggregates called lipofuscin and drusen, which are implicated in the pathogenesis of macular degenerations. Autophagy is a clearance mechanism that removes damaged cellular components, pathogens and other debris. Although autophagy is an emerging central player in the health and dysfunction of the RPE, we have limited insight into how this essential catabolic function is accomplished in the RPE and how it can be therapeutically targeted in intractable diseases like age-related macular degeneration. The goal of this research is to dissect the molecular regulation and execution of autophagy in the RPE in mechanistic detail. We will use high-speed live imaging of polarized primary adult RPE monolayers, gene disruption and the Abca4-/- mouse model of Stargardt disease to test specific hypotheses regarding how autophagic, phagocytic and lysosomal pathways intersect in the RPE and how this is affected by innate stressors like lipofuscin bisretinoid accumulation. The execution of autophagy can be divided into three phases that our Aims are directed to: initiation of autophagy (Aim 1), biogenesis of autophagosomes (Aim 2) and completion of autophagy (Aim 3). Aim 1 will test the hypothesis that outer segment phagocytosis inhibits the mechanistic target of Rapamycin complex 1 (mTORC1) in the RPE to activate the transcription factors TFEB and TFE3, which induce the expression of a comprehensive network of lysosomal and autophagy genes. Aim 2 will investigate the step-wise biogenesis and maturation of autophagosomes in real-time and test the hypothesis that outer segment-containing phagosomes are recruited into nascent autophagosomes as autophagic cargo. Microtubule-based transport of autophagosomes and lysosomes is required for degradation of autophagic cargo. Aim 3 will test the hypothesis that post-translational modifications of tubulin regulate the efficiency of autophagosome/lysosome transport and thereby control autophagic flux. These experiments will answer fundamental questions about cellular clearance in the RPE by addressing how autophagic-lysosome functions are scaled to meet the burden of outer segment degradation, how autophagic and phago-lysosomal pathways interact to maintain clean RPE, and whether autophagy promotes or prevents RPE dysfunction. Lack of mechanistic insight has significantly hampered therapeutic targeting of autophagy in retinal degenerations. We anticipate that this research will yield crucia information regarding autophagic clearance in the RPE and aid the development of strategies to preserve RPE health and function over the human lifespan.